1. Field of the Invention
The present invention relates generally to a method for producing particular products by recombinant microorganisms, and more particularly, to provide a method for producing biodegradable polymer and biomass fuel converted from carbon source by using recombinant microorganisms.
2. The Prior Arts
With progressing development of bio-diesel, using B2 bio-diesel blend as fuel of vehicles was promoted in Taiwan from 2000. However, the increasing of discarded crude glycerol accompanied with bio-diesel production results in environmental pollution. Although the discarded crude glycerol could be recycled and refined, the economic benefits are limited. Thus, treatment of this crude glycerol, byproduct of production, has becoming the key point for the development of bio-diesel. Regarding the trends of the world, most advanced countries have related laws made for energy policy, and majorities of which have being as principal items for national development. For example, the vehicles using petroleum must be replaced by those using substitute fuels with designate percentage in a given time; as well as increasing the percentage thereof gradually. Under those trends, the production quantities of bio-diesel will be increased relatively, and at that time it will certainly be a serious problem inasmuch as the enormous discarded crude glycerol. Therefore, if the crude glycerol can be efficiently processed and utilized by microorganisms, for example, producing biodegradable polyhydroxyalkanoate (PHA), the additional value resulted from bio-diesel production will be enhanced consequently. PHA, a kind of polyester existing in microorganisms, is generally served as a carbon and energy source. When the microorganisms are in a nutrients-limit condition, they will generate a hydrophobic inclusion body encompassing the PHA within the cell. The hydrophobic granules may have the PHA accumulation content over 90% of their dry weight under certain condition. Concerning the chemical structure, PHA is a kind of polymer composed of hydroxyl fatty acid, i.e., hydroxyalkanoic acid (HA); the structural formula thereof is presented as follows:
wherein the molecular weight range generally from several ten thousands to several millions, m is 1-4, n is 100-3000, and R is C1-C5. Variable kinds of PHA and monomers thereof differ in R group; in general, PHA compound comprising polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), and copolymer of PHB and PHV (i.e., linked both with PHB and PHV), etc. With different kinds of monomer composed, PHA may not only be a kind of hard and brittle plastics but also a soft elastomer, which involve a series of different characteristics thereof. PHA could be synthesized by using bin-renewable resources isolated from organisms as raw materials; moreover, it could be degraded by organisms, e.g., bacteria, when existing in natural environment, and hence could be a plastic substitute for conventional undegradable plastic. As a biocompatible and biodegradable thermoplastic polymer, it is suggested that PHA will have a good application in medical science and industry.
The production and development of PHA were started in the 1970s. ICI Company produced PHA by using microorganisms in the soil through fermentation process at that time. Meanwhile, Massachusetts Institute of Technology (MIT) introduced engineering techniques to produce PHA by using microorganisms. Owing to the research results, Metabolix Company was derived and established. The patents concerning the abovementioned results were assigned to Zeneca Company, and subsequently assigned to Monsanto Company. Metabolix Company, having purchased related patents from Monsanto Company in 2001, researched and developed on the basis of the techniques from patents and their own, and cooperated afterward with Archer Daniels Midland (ADM) to set up bulk scale production of PHA achieving 50 thousand tons in 2004 and intending to put to production in 2007-2008. The cost for PHA production is US$3-5/kg by fermentation method pertain to Metabolix Company; however, the cost may be reduced to less than US$2-3/kg by industrial process. Therefore, Metabolix Company planned to further abate cost to US$2-3/kg by improving innovative devices. Nevertheless, the highest part of cost is about the medium components employed for fermentation, which accounts for 50-80% of the operation cost. By the analysis of upstream and downstream material cost, the cost of carbon source and nitrogen source is more than 80% of total material cost. As the high cost of carbon source, commercialization and competition of PHA products are obstructed. Thus, if the crude glycerol produced by the bio-diesel factory could be recycled and utilized as a carbon source, it will be beneficial to achieve the objective of cost reduction for PHA production.
On the whole, the national research concerning PHA includes the strain improvement (e.g., Escherichia coli, Wauteria eutropha), gene mutation (e.g., genes of phaP, phaR), scale-up of culturing volumes, processes for fermentation production, tests for industrial application etc., what is more, the gene research and the strain improvements are both emphasized. In respect of Mainland China, which adopts biodegradable biopolymer material as the main issue for environmental protection of 2008 Beijing Olympic Games; meanwhile, the achievements regarding biodegradable plastics research are also planned to propagandize intensively. Besides, in Korea, the technique of high cell density fermentation for PHA production does have a referring value; moreover, the aspects of bacteria species refinement and the pathway improvement of metabolism also have been studied deeply. Furthermore, the German research group decodes the genomic DNA of Ralstonia eutropha, where the homologues genes of phaC, phaB, and phaA are found. It is not only a crucial breakthrough research for Ralstonia eutropha, but also could be applied to improve the production of other bacterial species by gene alignment and genetic engineering. In addition, various types of bio-polyester pertained to PHA, e.g., 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 4-hydroxybutyrate (4HB), are produced by using Bacillus cereus SPV from the research group of England.
Furthermore, the research group of Switzerland puts emphasis on using genetically modified plants, being different from microbiological expression system, to produce PHA. In U.S.A., other than the studies of bio-polyester production by means of recombinant E. coli expression system, the characteristics of copolymer are also analyzed so as to amplify the downstream application fields. Additionally, the research group of Australia deeply research on the gene regulation system of PHA production in bacteria pertained to Pseudomonas family, which indicates that the bacteria even of the same family present significant differences in gene level expression. Moreover, the Indian research group uses photosynthesis bacteria for PHA production; and Canada tests the biodegradability of PHA for further research.